Production of yeast biomass

ABSTRACT

A yeast biomass which is rich in intracellular RNA is produced by cultivating a microorganism of the genus Candida which is sensitive to potassium chloride. RNA has a wide application as condiments and pharmaceuticals.

United States Patent Akiyama et al.

14 1 Sept. 30, 1975 PRODUCTION OF YEAST BlOMASS Inventors: Shunichi Aki yama; Munebaru Doi;

I Yoshiaki Arai; Yoshio Nakao; Hideo Fukuda, all of Osaka, Japan Assignce: Takeda Chemical Industries, Ltd.,

Osaka, Japan Filed: Feb. 22, 1974 Appl.No.: 444,800

Foreign Application Priority Data Feb. 22, 1973 Japan 4841642 U.S. C1 195/28 R; 195/49; 195/82 1m. Cl. ClZB 1/00 Field of Search 195/28 R, 28 N, 37, 46,

Primary E.\'aminerLionel M Shapiro Assistant E\'aminerR. B. Penland Attorney, Agent, or Firn1Wenderoth, Lind & Ponack 57 ABSTRACT A yeast biomass which is rich in intracellular RNA is produced by cultivating a microorganism of the genus Candida which is sensitive to potassium chloride.

RNA has a wide application as'condimcnts and pharmaceuticals.

3 Claims, No' Drawings PRODUCTION OF YEAST BIOMASS This invention relates to a method for producing yeast biomass. More particularly, this invention relates to a method for producing yeast biomass rich in intracellular ribonucleic acid by cultivating a yeast of the genus Candida which is sensitive to potassium chloride and is able to assimilate at least one member selected from hydrocarbons, fatty acids, alcohols, oils and fats.

A remarkable demand has recently been observed in the industrial fields of nucleic acid utilization which, starting with ribonucleic acid (hereafter briefly RNA), produces '-nucleotides such as 5'-inosinic acid, 5'- adenylic acid, 5-guanylic acid, 5'-cytidylic acid, 5'- uridylic acid, etc., which have been widely used as condiments, pharmaceuticals or their intermediates, and the corresponding nucleosides also have an increase demand as a material for RNA. This, in turn, has made it an important goal to manufacture RNA at low cost and in large quantities.

Heretofore, RNA has been harvested mainly from the biomass obtained by growing a yeast, particularly a yeast of the genus Candida in a culture medium containing molasses or pulping waste liquor as a principal source of carbon.

In this connection, several methods have been proposed for securing yeast biomass rich in RNA and, from technical points of view, these methods can be classified into the following three groups, namely: Group 1: Methods for obtaining yeastbiomass rich in RNA in which yeasts are grown under specified cultural conditions.

Group 2: Methods for obtaining yeast biomass rich in RNA in which yeasts are grown in a medium supplemented with some agents with specified activities or some precursors.

Group 3: Methods for obtaining yeast biomass rich in RNA, wherein the yeasts to which specified genetic features have been imparted are cultivated. However, the methods classified in Group 1 not only require specificfermenters and ancillary equipment, but often encounter considerable difficulties in completing the fermentation. The methods of Group 2 require the use of some specified agents or precursors in which expensive materials or toxic substances such as heavy metals are mostly used which necessarily require troublesome procedures for avoiding possible pollution problems in the treatment of the fermentation waste liquor.

Furthermore, the methods of Group 2 are not desirable from the standpoint of public hygiene in that the RNA of yeast biomass containing such'heavy metals or other toxic substances, either adhered or adsorbed thereto, is to be used as a starting material in the production of condiments, pharmacenticals or their intermediates.

Therefore, as possible means for producing yeast biomass rich in RNA, the art belonging to Group 3 is most desirable. However, the only known method of this type is the one involving the use of polyploids of Saccharomyccs yeasts and yet even this method has not up to now provided sufficiently high yields.

Under the circumstances the present inventors conducted an intensive study to find yeasts which are able to utilize less expensive carbon sources and yet to give a biomass rich in RNA. The study led to thefinding that yeasts belonging to the genus Candida, which are sensitive to potassium chloride,-are extremely rich in RNA.

The finding gave the impetus to thedevelopment of this invention.

Thus, the main object of the present invention is to 5 provide a method for producing a yeast biomass which is rich in RNA. 1

Another object of this invention is to provide a process for producing RNA in a high yield.

Further object will be explained in the ensuing description.

The yeasts of the genus Candida which are to be employed in working this invention include, among others, Candida albicans, Candida bovina, Candida brumptii, Candida catenulata, Candida claussenii, Candida curiosa, Candida curvata, Candida diddens, Candida diversa, Candida glaebosa, Candida guilliermondii, Candida humicola, Candida intermedia, Candida kefyr, Candida krusei, Candida lambica, Candida lip0lytica(This includes the yeasts classified as Saccharomycopsis lipolytica by Yarrow in Antonie von Leeuwenhoek 38 357360( 1972)), Candida Iusitaniae, Candida macedoniensis, Candida melinii, Candida membranaefaciens, Candida mesenterica, Candida moggi, Candida norvegensis, Candida parakrusei, Candida parapsilosis, Candida pelliculosa, Candida pseudotropicalis, Candida panicea, Candida reukaufii, Candida rugosa, Candida sake, Candida salmonicola, Candida slooffii, Candida solani, Candida stellatoidea, Candida tennis, Candida tropicalis, Candida utilis, Candida valida, Candida vini, Candida zeylanoides, etc.

The yeasts which are referred to in this specification as sensitive to potassium chloride are those which comply with the following conditions: (An index to the determination of the yeasts sensitive to potassium chloride) A loopful of cells are picked up from a slant culture of a given yeast of the genus Candida and inoculated into a test tube containing 3 ml. of medium A (described hereafter). The inoculated medium is incubated under shaking at 28C for 24 hours and 0.025 ml. of the resultant culture is transferred to a test tube containing 3 ml. of Medium B (described hereafter), in which the yeast is shake-cultured at 28C for 48 hours. The resultant culture is diluted to 1/50 concentration with water and a sample is put in a cell, 16 mm in inner diameter. The sample is measured for absorbance at 590 mg. by means of a spectrophotometer. When the absorbance value is not more than 0.050, the particular yeast tested is judged to be sensitive to potassium chloride. As a spectrophotometer for this purpose, Coleman Junior Spectrophotometer Model 6D, for instance, can be employed to advantage.

Medium A: 3 percent of glucose, 0.3 percent of meat extract, 0.3 percent of yeast extract, 0.5 percent of urea and 0.01 percent of FeSO,,.7H O. It should be understood that when the yeast to be tested is not able to utilize glucose as carbon source or requires some special nutrient, one may employ Medium (A) which contains, in place of said glucose in Medium A, other carbon source or which is supplemented with some nutrients which the particular yeast requires for its growth. Medium B (or Medium B) is equivalent of Medium A V (or MediumjA to which potassium chloride has been addedin a molar concentration of 1.5. Such Candida'ye'asts sensitive to potassium chloride can be easily selected by the so-called replica-plating method,'in which each Candida yeast to be tested is grown-on Plate Media Aa(A'a) and Ba(-B'a), which are Medium A(or A) and Medium B(or B), to which agar has been added, respectively, why the procedure set out above as an index to.the determination of yeasts sensitive to potassium chloride. In this connection, it is advisable to use'a yeast of the genus Candida as a par ent strain, to treat it with, for example, ultraviolet rays, X-rays or with chemicals such-as Nmethyl-N-n'itro-N- nitrosoguanidine, quinoline, nitrogen mustard, hydrogen peroxide'or'dimethylsulfoxide to induce mutation and to single out a suitable strain that satisfies the above index by employing, for example, the replicaplatingmethod mentioned above.

Among these potassium chloride-sensitive strains, there are obtained yeasts whose biomass contain intracellular RNA as high as- 12 percent. To measure the RNA contentof the biomass of such a strain, the strain is cultivated under the following medium and cultural conditions, for instance, and the RNA contents of the resultant yeast biomass are measured.

In this case by using a less expensive material as a carbon source of the media, such as any of hydrocarbons, acetic acid and alcohols, for instance, strains which are able to assimilate such carbon sources can be simultaneously selected. A typical selection procedure is as follows. (Experiment) RNA Content 28C on a rotary shaker at 200 r.p.m. for 16 to 24 hours. To the culture broth thus obtained is added a single drop ofa surface active agent (e.g. Emulgen 420 (Kao Atlas, KK, Japan) 4. Method of analysis 1. Determination of the amount of yeast biomass Five milliliters of the culture broth is centrifuged at 3000 r.p.m. for 15 minutes to harvest the biomass, which is then dried at 105C for 24 hours. The dry weight of the biomass is taken as the amount of the biomass.

2. Measurement of RNA content A 1 ml. portion of the culture broth is taken and its RNA content is measured by the method of Schmidt G. and Thannhauser, S. (Journal of Biological Chemistry 161, 83 (1945)). The amount thus found is expressed in terms of percentage per unit weight of biomass.

Emergence of yeasts which give a biomass rich in RNA ("/1) 0-2 4 -6 8 -10 -l2 l4 -l6 l8 2() Total Group A 2 195 3 200 strains ,Group B 83 114 2 199 1 strains Group C X8 102 7 2 7 4 210 strains l. The strain to be tested: Candida'lipol'ytica TA-540,

lFO 1657 (ATCC) 2. Method for isolating strains (Group A) A cell suspension of the test organism (0.1 M phosphate buffer, pH 7.0) is diluted to a suitable concentration and the diluted suspension is smeared onto Plate Medium Aa (described above).

After 2 day cultivation at 28C'200 strains are isolated at random from among the colonies developed. (Group B) To a cell suspension of the test organism (The same buffer as above) is added a sufficient amount of N-methyl-N-nitro-N-nitrosoguanidine to give a concentration of 300 #g/ml. and after a contact time of 30 minutes at 28C, the cells are washed twice with sterile water and smeared onto Plate Medium Aa, which is incubated at 28C for 2 days. From the resultant colonies there were picked up 199 strains at random. (Group C) The test organism is treated in entirely the same manner as that set forth above for Group B and,'by means of the replica-plating method described hereinbefore for the selection of potassium chloridesensitive organisms, 210 potassium chloride-sensitive mutants are isolated.

3. The method of growing the isolate d strains For all of the above groups, eachofvthe isolated strains was cultivated in the following manner. A loopful of yeast cells, picked up from a slant culture are used to inoculate a 200 ml. creased conical flask containing 20 ml. of Medium S (see Example.1).

The present inventors have been studying the reason for the high RNA contents of potassium chloridesensitive yeasts but the phenomenon has not been completely accounted for as yet. However, it appears that potassium chloride bears a close relationship with the intracellular biosynthesis of RNA or metabolism controlling mechanisms of Candida yeasts. In the present specification and claims, yeast biomass which is referred to as rich in RNA is the one containing intracellular RNA not less than 12 percent, preferably 14 percent, and most desirably 15 percent.

The following method serves as a practical index evaluating the richness of biomass in RNA. Namely, to 0.5 ml. of a cultured broth, there is added 5.0 ml. of a mixture of ethanol-ether (1:1 and the whole system is stirred well, which is then subjected to centrifuge at 2,000 r.p.m. for 5 minutes to obtain precipitates. The resultant precipitates are washed with a 0.2N aqueous solution of perchloric acid and mixed with 1 ml. of a lN aqueous solution of sodium hydroxide. After the mixture is maintained at 37C for 20 hours, it is filled up to 10 ml. with a 10 percent aqueous solution of perchloric acid and filtered. The resultant filtrate is diluted ten times with a 0.2N aqueous solution of perchloric acid. The optical desitity of the diluted solution at 260 mp.(c=1.0 cm) is measured and expressed as A.

On the other hand, 5 ml. of said cultured broth is subjected directly to centrifuge at 3,000 r.p.m. for 15 minutes to obtain precipitates, which is dried at C for 24'hours, the dry weight (milligram(s) of the precipitates is expressed as B.

Thus, in case where C in the following equation isnot less than 0.12. this indicates that the tested biomass is rich in-RNA-.

In accordance with this invention, a yeast of the genus Candida having a sensitivity to potassium chloride and being able to give a biomassrich in RNA is cultivated under aerobic conditions. The carbon sources that can be employed include, among others, any one, or a mixture of two or more of various hydrocarbons such as'decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, eicosane, kerosene, gas oil, heavy gas oil, etc., fatty acids such as acetic-acid,

propionic acid, butyric acid, myristic acid, margaric acid, palmitic acid, oleic acid, etc., oils and fats'such as soybean oil, sperm oil, cottonseed oil, etc., and monohydric and polyhydric alcohols such as methanol, ethanol, propanol, glycerol, etc. In particular, it is advantageous, from economic points of view, to employ the carbon sources which are available at low cost and in large quantities such as the primary and secondary products of petrochemical industries.

The nitrogen sources which can be employed to advantage in the cultivation according to this invention include organic nitrogeneous sources such as corn steep liquor, cottonseed cake, yeast extract, fish meal, corn meal, etc., inorganic ammonium salts andnitrates such as aqueous ammonia, ammonia gas, ammonium sulfate, ammonium chloride, ammonium carbonate, ammonium phosphate, sodium nitrate, ammonium nitrate, etc., and organic compounds such as urea and amino acids. i x.-

In addition to these sources of carbon and nitrogen, the medium is supplemented with various minerals adequate to the growth of the yeast to be employed, such as salts of iron, manganese, calcium, zinc, copper, aluminum, etc., phosphoric acid, etc., as well as vitamins and amino acids which may be required for their growth. The fermentation can be easily conducted continuously or batchwise. v v

The pH of the medium to be employed and of the culture broth in the course of cultivation is maintained simply within the optimum growth pH range of the yeast to beemployed which is usually between about pH 2 and about pH and'preferablybetween about pH 3 and pH about 7.5. I

To maintain the pH of the culture ,broth during the cultivation within such an optimum range, the medium may be supplemented, from time to time, with hydrochloric acid, sulfuric acid, phosphoric acid, aqueous ammonia, ammonia gas, an aqueous sodium hydroxide solution, an aqueous sodium carbonate solution, calcium carbonate, calcium hydroxide or the like or such organic acids as acetic acid.

Particularly, a solution containing suitableproportions of acetic acid on the one handand aqueous ammonia, ammonia gas or aqueous sodium hydroxide solution on the other hand not only serves asboth-carbonand nitrogen sources but also functions to control the pH and, therefore, such a solution as. above is particularly desirable for the cultivation of these yeasts from economic points of view as well.

1 As to the. cultivation temperature, the temperature suited to the growth and intracellular accumulation of RNA of the particularstrain is selected; usually from the range of about 10C to 40C. Preferably, the, cultivation is'made at about 15C to 28C, and thecultivation period should be so selected that canprovide a high'RNA content economically, which may of course be widely variable with the medium'employed as well as other cultural conditions.

The procedures for harvesting yeast biomass richin RNA from the thus obtainedculture broth containing the yeasts which aresensitive to potassium chloride and which is rich in RNA' including for example a continuous or batchwise centrifugal separation method, filtration, sedimentation and other routine methods employable for the separation of microbial biomass.

The invention will be described in further detail by examples. I I

The IFO numbers assigned to the microorganisms used in the present specification indicate the accession numbers at the Institute for Fermentation, Osaka, Ja-

The ATCC numbers assigned to the microorganisms used in the presentspecification indicate the accession numbers at American Type Culture Collection, 12501 Parklawn Drive Rockville, Maryland 20852, U.S.A.

The quantitative determinations of RNA were performed by the method of Schmit, and Tannhauser, S Jour,nal (mentioned before).

In the following examples, the relationship between part('s)' by weight and part(s) by volume corresponds to 'that between gram(s) and millilitre(s).

EXAMPLE 1 Candida lipolytica IEO I657 (ATCC) was treated withN-methyl-N'-nitro-N-nitrosoguanidine in the conventional manner and the mutants were screened by the replica-platingmethod described above. 'Mutant 'L-42 (IFO 1648; ATCC 20400) thus selected was culti- The absorbance found was 0.020, The absorbance for Candida lipo lytica IFO 1657'(ATCC) which had been cultivated in the same manner was 0.188. Then, the yeast cells were takenfrom a slant culture of strain L-42 and inoculated into a fermenter containing parts by volume of medium S ,(described hereafter), The cultivation was conducted at 28C for 24 hours. 20 parts by volume time resultant culture was transferred to a fermenter containing 1,000 parts by volume of a medium which was basal medium M.(describecl hereafter) supplemented with the varying kinds of carbon sources indicated in Table I, wherein the organisms were grown at the temperature of 28C and the agitator speed of 1,500 r.p.m., with sparging at the rate of 1,500 parts by volume/min. Throughout this period, a 25 percent aqueous solution of ammonia was automatically fed so as to maintain the pH of the medium in the neighborhood of 4.5. Separately, cells of Candida lipo- Jytic-a'IFO 165 7 (ATCC) were picked up from a slant culture thereof in a similar manner and cultivated by a procedure similar to that described above.

.The amount'of yeast biomass and the RNA content of the biomass in each of the above cultures were measured. ,The results are, shown in Table l.

To harvest RNA from the thus-obtained yeast biomass rich in RNA, conventional separatory procedures could be applied. A typical procedure was as follows. Thus, 100 parts by weight of these biomasses were sus- (ATCC) was also grown by precisely the same manner as above. The amount of biomass and RNA content of each of these culture broths were measured by procedures similar to those set forth in Example l. The repended in 1000 parts by volume of a 10 percent solun a given in Table 2. tion of ammonium sulfate and the suspension was Table 2 heated at 90C for 4 hours to extract the RNA. After cooling, the system was centrifuged and the supernapurer" 5min Strain L42 tant was brought to pH 2.0 by the addition of hydro- Cultivation Dry Wcigm RNA Dry weight RNA chloric acid. The resultant crude RNA precipitates time f iomass ontent of biomass content were collected by centrifuge, washed w1th 60 percent '8 hrs (mg/L HA7 5,6 g". T69! ethanol and dried under reduced pressure. The proce- 15.5 9,9" 8.9 17.5" dure yielded 10.4 parts by weight of crude RNA pow- 22 12.7 17.9" 24 14.9 17.8" der.

Table l Candida lipalylicu Candida lipolyliva [F0 1657 (ATCC) 1F0 I648 (ATCC 20400 (parent strain) (mutant strain) Carbon source Cultivation Dry weight RNA Cultivation Dry weight RNA (Concentration) time of biomass content time of biomass content G1 cer61 2% 16 hrs. 10.0 g/l 10.0% 18 hrs 9.1 g/l 17.4% Ethanol 1% 16 6.7 9.6" 16 6.3 17.0" Acetic acid (l7!) l8 4.6 9.3 l8 4.4 17.8" Soybean O 2% 20 [8.5 8.0" 22 16.3 15.0" n-Paraffin (2% 20 21.0 9.1" 22 18.1 17.5"

(Commercial mixture.

0.003% of FeSO .7H O, 0.01% of CaCl 2H O. 0.l'7( of NuCl. 0.3% of yeast extract, 0.3% of corn steep liquor and 0.01% of Actocol.

EXAMPLE 2 A slant culture of L-42, lFO 1648 (ATCC 20400 a potassium chloride-sensitive mutant induced from Candida Iipolytica lFO 1657, was used to inoculate the first seed fermenter containing 500 parts by volume of medium S (See Example 1) and cultivated under shaking at 28C for 24 hours. The entire amount of the resultant culture was used to inoculate the second seed fermenter containing 25,000 parts by volume of medium S and cultivated at 28C for 24 hours. 2,000 parts by volume of the culture thus obtained was transferred to a main fermenter containing 100,000 parts by volume of a medium, which was the basal medium M of Example 1 supplemented with l.5 percent (weight- /volume) of n-hexadecane and cultivated at 22C under aeration for about 24 hours. Throughout this period, percent aqueous ammonia was automatically fed so as to maintain the pH of the medium in the neighborhood of 4.5. Separately, Candida lipolytica lFO-1657 The n-hexadecane in the medium was exhausted within 20 hours in the case of the parent strain and in 24 hours in the case of L-42.

EXAMPLE 3 A strain of Candida tropicalis, NH-20, lFO 1649 (ATCC 20401), which has been isolated from a soil sample obtained in the suburb of Nara, and strain lFO 1400, which is known as a standard strain (type culture) of this species, were each cultivated and the absorbance of each culture was measured by the abovedescribed assay procedure for potassium chloride sensitivity. The results are 0.025 for the former and 0.166 for the latter. Then, the two strains were grown in the same manner as Example I (provided that the cultivation temperature in basal medium M supplemented with various carbon sources was 22C). The amount of biomass and intracellular RNA content of each of these biomasses are shown in Table 3.

Table 3 Candida lropicalis Candida rrupiculix lFO l649 (ATCC 2040l) lFO l400 Carbon source Cultivation Dry weight RNA Cultivation Dry weight RNA (Concentration) time of biomass content time of biomass content Acetic acid (l7!) l8 hrs. 44 g./l. l6.294 18 hrs. 4.2 g./l. 9.8% Glycerol (2%) l8 hrs. 9.0 g./l. I097! 18 hrs. 8.4 g./l. 9.671 n-Paraffin (271) 22 hrs. 20.0 g./l. 16.771 22 hrs. 2l.l g./l. 9.3V! (mixture.

EXAMPLE 4 Table 6 Continued Test strain Absorbance Candida alblcans [F 1650 (ATCC 20402), WhlCh had been isolated from a soil sample collected in the ('amlida kruxui IFO 0592 0.220 suburb of Osaka, and Candida guilliernwndii lFO 1651 5. Wa -g (ATCC as as the Standard Strains yp Candida parapxilusix [F0 0708 A 0.230 cultures) of these species, i.e., Candida albicans lFO Pam/18mms (W15 I IFO 0566 (ATCC 20406 1060 and Candida gmllzermondn were re- (mm/u "mum ":0 m0 um) spectively cultivated and theabsorbances of the resul- Cam/i111: rvhus/u R415 IF 1655 40 (ATCC 20407) cultures w j ff by the prfjcedpre 0 Candida .rloI/amidaa lFO 1399 0.125 scnbed above in the definition of potassium chloride Candida .m'llamidea 5-541 0.030 sensitivity. The results are set forth in Table 4. I '2% Then, each of these strains was cultivated in the same 7 manner as Example 3 and the amount Of Cells nd e Then, these strains and, by way'of controls, their parlular RNA content of the resultant culture were deterent strains were each cultivated by the procedure of mined. The results are set forth in Table 4. Example 3 in which acetic acid and n-paraffin were used as carbon sources.

The amount of biomass and intracellular RNA content of the biomass were measured. The results are Table 4 shown in Tables 7 and s.

TABLE 7' Test strain -Absorbance Candida albicans 11=o 1650 (ATCC 20402) 0.045 Test Strain cultivfllinn y ht RNA Candida gliilliernmndii lFO 1651 (ATCC 20403 0.030 "fbmmflss Candida albit'am' lFO 1060 0.125 Candida guillicrmundii [F0 0566 0150 Candida lnlarnmdia 20 hrs. 3.9 g./1. 7.87!

Table 5 Carbon source Test strain Cultivation Dry weight RNA (Concentration) time of biomass content Candida albic'ans IFO 1650 20 hrs. 4.0 g./I. 15.3'7:

(ATCC 20402 Acetic acid lZ') Candida albicans [F0 1060 20 hrs. 4.3 g./l. 8.3% Candida guilliermundii IFO 1651 18 hrs. 4.4 g./l. 15.5'71

(ATCC 20403 Candida guilliermondii IFO 0566 18 hrs. 3.9 g./l. 7.771 Candida albicans [F0 1650 26 hrs. 16.) g./l. 15.8%

(ATCC 20402) n-Paraffin (2'7!) Candida albicans IFO I060 26 hrs. 16.0 g./l. 7.871 (mixture. cp -CH1) Candida guilliermondii lFO 1651 22 hrs. 18.3 g./l. 15.4%

(ATCC 20403 Candida guilliernwndii lFO 0566 22 hrs. l8.9-g./l. 8.5%

EXAMPLE 5 Candida inlemledia 20 hrs. 4.0 g./l. 15.07! 1-330 lFO 1652 Candida lntermedza IFO 0761, Candida krusez IFO (ATCC 20404) 0592, Candida parapsilosis IFO 0708, Candida robusta i 0592 Candida [\I'llSt'l 18 hrs. 4.l g./l. 15.7% [F0 1000 and Candida stellatozdea IFO 1399 were each 45 K409 1653 treated with N-methyl-N '-nitro-N-nitrosoguanidine I C 2 and screened by the replica-plating method described i'gg 0708 20 hereinbefore. The mutant strains thus selected, Can- Candida parapsilasis 20-hrs. 4.2 g./1. 15.2% dida intermedia 1-330 (lFO 1652) (ATCC 20404 Candida krllsei 9 F I653)(ATCC Candida Candida rubnxla 20 hrs. '1 3.7 g./1. 11.1% parapsilosis P-74 (lFO 1654)(ATCC 20406), Candida 0 9.

' Candida robusla 20 hrs. 3.6 g./l. 16.07r robusta R-l and Candida R 15 ":0 I655 j stellatoidea S-54l (IFO 1656) (ATCC 20408), Iwere (A CC 2 07) r each cultivated and the absorbances of the resultant igs H99 20 cultures were measured by the procedure set out in the 55 Candida srellaloidea 20 hrs. 4.4 g./l. 15.5% index of the determination of potassium chloride sensi- (ATCC 20408 tivity.

The results are set forth in Table 6. As controls, their Cur nn wurct-vacctic acid. 1.0% parent strains were also cultivated in the same manner and thc absorbances of the resultant cultures were simi- Table 8 larly measured. The results were as indicated below.

, Test strain Cultivation Dry weight RNA Table 6 time of biomass content Test ruin Absorbance 5 Candida inlermcdia 26 hrs. 14.0 g./l. 7.9% IFO 0761 Cam/Ill" lt'diu 11:0 0761 0.1 15 Candida inu'rmedia 26 hrs. 13.5 g./l. 15.27: Candida inlarme'dia L330 lFO 1652 0.040 1-330 [F0 1652 (ATCC 20404) (ATCC 20404) Table 8-C'ontinucd Carbon source: n-pnrnffin (mixture (il'C -(mk 27:

Test strain Cultivation .Dry weight RNA time 7 of biomass content Candida krurrri lFO 0592 22 hrs. 17.9 g /1 7.7% Candida krusci 22 hrs. 16.5 /1 15.07!

-K- 1091FO1653 (ATCC 20405) Candida purapsi/mis 24 hrs. 1.71% g./l. 8.371

[F0 0708 I Candida parapxilusix 26 hrs 18.0 g./l. 15.17:

P-74 lFO 1654 (ATCC 20406) Candida mbuxlu v 26 hrs 153 g./l. 7.971

lFO-1000 Candida rohuxlu 26 hrs 15.4 g./1. 15.971

. R -115 lFO'1655 1 (ATCC 20407). v H Candida .m'llalbidea i 22 hrs l8.3'g./l. 6.4%

lFO 1399 Candida xlellamideu 22 hrs 17.0 g./ lv 15.5%

What we claim is:

l. A method for producing yeast biomass which comprises; cultivating a yeast of the genus Candida, which is sensitive to potassium chloride and is able to assimilate at least one member selected from the group consisting of hydrocarbons, fatty acids, alcohols, oils and fats, in a medium containing at least one of said assimilable carbon sources as a principal carbon source; thereby producing in the cultivated medium the yeast biomass which contains not less than 12 percent by weight of intracellular ribonucleic acid; and recovering said biomass therefrom.

2. A process according to claim 1, wherein said yeast of the genus Candida is one belonging to any of the species Candida lipolytica, Candida tropicalis, Candida guilliermondii, Candida albicans, Candida intermedia, Candida krusei, Candida parapsilosis, Candida robusta, and Candida stellatoidea.

3. A process according to claim 1, whefein the resulting yeast biomass contains ribonucleic acid in amounts not less than 14 percent by weight. 

1. A METHOD FOR PRODUCING YEAST BIOMASS WHICH COMPRISES, CULTIVATING A YEAST OF THE GENUS CANDIADA, WHICH IS SENSETIVE TO POTASSIUM CHLORIDE AND IS ABLE TO ASSIMILATE AT LEAST ONE MEMBER SELECTED FROM THE GROUP CONSISTING OF HYDROCARBONS, FATTY ACIDS, ALCOHOLS, OILS AND FATS, IN A MEDIUM CONTAINING AT LEAST ONE OF SAID ASSIMIABLE CARBON SOURCES AS A PRINCIPAL CARBON SOURCE, THEREBY PRODUCING IN THE CULTIVATED MEDIUM THE YEAST BIOMASS WHICH CONTAINS NOT LESS THAN 12 PERCENT BY WEIGHT OF INTRACELLULAR RIBONUCLEIC ACID, AND RECOVERING SAID BIOMASS THEREFROM.
 2. A process according to claim 1, wherein said yeast of the genus Candida is one belonging to any of the species Candida lipolytica, Candida tropicalis, Candida guilliermondii, Candida albicans, Candida intermedia, Candida krusei, Candida parapsilosis, Candida robusta, and Candida stellatoidea.
 3. A process according to claim 1, wherein the resulting yeast biomass contains ribonucleic acid in amounts not less than 14 percent by weight. 